W4L3 IVF and assisted reproductive technologies Flashcards
Definition of infertility
- Unprotected intercourse for 1 year without conception
Assisted Human Conception
§1 in 6 couples will require medical intervention to conceive
§Around 5% of all children born in Australia are so- called ‘test-tube’ babies
§There are over 10 million test-tubes babies world-wide
Endometrial receptivity
window of implantation b/w 19-23 days post ovulation allows embryo to attach
Age- related decline in human female fertility
- After 20s, the chance of conception drop sharply
The Paradox of Infertility and Multiple Gestations
- In IVF, transfer more than one fertilized egg to ensure that one will develop
-lead to twins , even triplet
Risk to mother in twin/ trip
§ Hypertension
§ Thrombo-embolism
§ Urinary tract infection
§ Anemia
§ Placental abruption
§ Emergency Caesarean section
§ Increased mortality rate
Risk to children in twins
-higher chance of cerebral palsy
-higher mortality
-cost of prematurity
-also social impact (divorce)
IVF Process
§ Stage 1: follicles are stimulated using exogenous gonadotrophins
Ø Exogenous FSH causes 6-20 follicles to be recruited + develop simultaneously
§ Stage 2: mature eggs are removed from ovaries
Ø LH/hCG triggers ovulation + eggs are removed prior to ovulation
Ø Oocytes collected by inserting needle into follicle + aspirating contents using local anaesthetic + ultrasound
§ Stage 3: eggs are fertilised with sperm (100,000/mL/egg)
Ø Intra-cytoplasmic sperm injection (ICSI): sperm injected directly into egg past cumulus + zona
§ Stage 4: fertilised embryos are grown in the laboratory + subsequently replaced into uterus
§ Stage 5: any embryos not transferred can be cryopreserved
Embryo Culture
Pronucleate oocytes are cultured in groups of 4 in 50μl drops of Phase I medium under paraffin oil for 48 h until Day 3
6% CO 2 , 5%O 2 , @ 37 o C.
Blastocyst Culture
-On Day 3, embryos are moved to 50μl drops of Phase II medium under oil for 48h to the blastocyst stage (Day 5)
-6% CO 2 , 5%O 2 , @ 37 o C.
Important to know in life egg transplant
-Oocyte is the determination variable in women fertility
-lower chance as the age is higher
Source of genetic material for genetic analysis
§ Polar body biopsy: extracted + analysed to determine maternally derived aneuploidies, no info on paternal genome
§ Blastomere biopsy: acid solution to make a hole in zona + remove a blastomere
§ Trophectoderm biopsy: not removing any embryonic tissue, can remove more than one cell for accurate diagnosis
Ø Genetic analysis: SNP array, qPCR or NGS (whole chromosome analysis, detects segmental aneuploidies)
Physiology Analysis
§ Metabolomics/proteomics: embryo consumption + production can be measured
Ø Healthy embryos are more metabolically active than unhealthy embryos
§ Females are more metabolically active as many metabolic genes are found on X chromosome
Fanconi Anaemia (FA) case study
autosomal recessive disease that results in inability to repair DNA damage
Ø Leads to congenital defects + numerous cancer types (leukemia)
§ Diagnosis: chromosome breakage test
Treatment: bone marrow transplant – cells from patient’s bone marrow replaced with someone else’s cells
Ø Cells from donor bone marrow or umbilical cord blood can serve as source of transplant
Ø 2 year survival rate following BMT 60-85% with matched sibling donor, 20-40% with donor from bank
-IVF sibling for match
Cryoperservation
§ Dehydration: cryoprotectant placed in base medium extracts water from cell
§ Slow freezing (hours): low levels of cryoprotectants → slow controlled rates of cooling (0.3˚C/min) → slow dehydration of cells to minimise ice crystal formation + damage
§ Vitrification (seconds): high levels of cryoprotectants → extremely fast rates of cooling (20,000˚C/min) → no ice crystal
formation/damage into glass-like structure
